Carnosic Acid Protects against Bisphenol A-Induced NLRP3 Inflammasome Activation by Attenuating Oxidative Stress in Human Microglial Cells

TL;DR

Carnosic acid from rosemary reduces BPA-induced inflammation and oxidative stress in human microglial cells.

Contribution

This study shows carnosic acid protects against BPA-induced NLRP3 inflammasome activation via antioxidant mechanisms.

Findings

CA reduced BPA-induced NLRP3 fluorescence and cleaved caspase-1 in microglial cells.

CA attenuated BPA-induced oxidative stress and restored antioxidant enzyme expression.

CA's protective effects were blocked by BSO, indicating a glutathione-dependent mechanism.

Abstract

Bisphenol A (BPA) is an endocrine-disrupting chemical commonly found in consumer products and is known to induce neuroinflammation and oxidative stress via microglial activation. Carnosic acid (CA), a phenolic diterpene from rosemary (Rosmarinus officinalis L.), has potent antioxidant and neuroprotective properties. This study investigated the protective effects of CA against BPA-induced neuroinflammation and oxidative stress in human HMC3 microglial cells. Firstly, BPA induced the expression of NLRP3 inflammasome–related proteins and increased NLRP3 fluorescence intensity. CA reduced the fluorescence intensity of NLRP3 and cleaved caspase-1 induced by BPA. CA also attenuated BPA-induced protein levels in the inflammasome, proinflammatory cytokines, and phosphorylated tau, as well as the transcription factors FoxO1 and p65. CA improved the effects in BPA-induced ROS level and reduced the expression of catalase, glutathione reductase, superoxide dismutase 2 (SOD2), glutathione peroxidase 1, heme oxygenase-1, nuclear factor erythroid 2-related factor 2 (Nrf2), and UDP-glucuronosyltransferase 1A1 (UGT1A1). In addition, CA restored BPA-suppressed expression of UGT1A1 and SOD2 in the striatum of mice. L-Buthionine-sulfoximine (BSO) treatment abolished CA’s ability to counteract BPA-induced oxidative stress and inflammatory responses. These findings indicated that CA was associated with attenuation of BPA-induced NLRP3 inflammasome-related protein expression and downstream neuroinflammatory signaling by reducing oxidative stress in HMC3 cells.

Figures

BPA induced the protein expressions of NLRP3 and ASC in HMC3 cells. (A) HMC3 cells were treated with 0.1% DMSO or 2, 20 and 200 nM BPA for 3 h. (B) HMC3 cells were treated with 0.1% DMSO or 20 nM BPA for 1, 3 and 6 h. The protein expression was measured by Western blotting. GAPDH was used as the internal control. The protein level in control was regarded as 1. (C) HMC3 cells were treated with 0.1% DMSO or 20 nM BPA for 3 h. The green fluorescence indicated NLRP3. The nucleus is stained with Hoechst 33258 (blue). The merged images show the overlap of both fluorescences. The images shown are rep

CA improved BPA-induced the proteins of NLRP3 and cleaved-caspase1 in BPA-treated HMC3 cells. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA for 3 h. (A and B) The green fluorescence indicated NLRP3 and cleaved-caspase1. The nucleus is stained with Hoechst 33258 (blue). The merged images show the overlap of both fluorescences. The images shown are representative from three individual. (C) HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 3 and 24 h. IL-1β production was measured by ELISA. (D) Cell viability was measured by MTT assay. The level i

CA improved BPA-induced the protein expressions in inflammasome, pro-inflammatory cytokines and inflammatory related proteins in HMC3 cells. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 3 h. The protein expression was measured by Western blotting. GAPDH was used as the internal control. The protein level in control was regarded as 1. Values are shown as mean ± SD (*n* = 3 biological triplicates). p* < 0.05, *p* < 0.01, p* < 0.001 compared to control group. ^#^*p* < 0.05, ^##^*p* < 0.01, ^###^*p* < 0.001 compared to BPA group

CA improved HMC3 cells against BPA-induced oxidative stress. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 0.5 h. (A) Reactive oxygen species (ROS) production was measured by flow cytometry. Fluorescence signals represent ROS production. (B) ROS generation was determined using DCF-DA assay. ROS level was visualized as DCF-DA green fluorescence. Values are shown as mean ± SD (*n* = 3 biological triplicates). p* < 0.05, p* < 0.001 compared to control group. ^#^*p* < 0.05, ^###^*p* < 0.001 compared to BPA group

CA reversed the BPA-decreased protein expressions in antioxidant enzymes and Nrf2 in HMC3 cells. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 3 h. The protein expression was measured by Western blotting. (A) GAPDH was used as the internal control. (B) PARP was used as the internal control. The protein level in control was regarded as 1. Values are shown as mean ± SD (*n* = 3 biological triplicates). *p* < 0.01, p* < 0.001 compared to control group. ^#^*p* < 0.05, ^##^*p* < 0.01, ^###^*p* < 0.001 compared to BPA group

CA reversed the proteins related to the antioxidant and detoxification enzyme in BPA-induced mice and HMC3 cells. (A) Mice were divided into 4 groups: control (olive oil 0.01 ml/g weight), BPA (50 µg/kg BPA), CA 5 (50 µg/kg BPA + 5 mg/kg CA), and CA 20 (50 µg/kg BPA + 20 mg/kg CA). (B) HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 3 h. The protein expression was measured by Western blotting. GAPDH was used as the internal control. The protein level in the control was regarded as 1. Values are shown as mean ± SD (*n* = 3 biological triplicates). p

BSO blocked CA reversed the ROS and protein levels of antioxidant enzyme, NLRP3 inflammasome and inflammatory-related proteins in BPA-treated HMC3 cells. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 0.5 and 3 h. Cells were pretreated with 100 µM BSO for 18 h before treatment with CA and BPA. (A and C) The protein expression was measured by Western blotting. GAPDH was used as the internal control. The protein level in control was regarded as 1. (B) ROS production was measured by flow cytometry. Fluorescence signals represent ROS production. Value

Full text

Introduction

Bisphenol A (BPA) is an environmental endocrine-disrupting chemical (EDC) widely present in plastic products, water bottles, and can linings. Chronic exposure to BPA has been associated with adverse effects on the nervous, cardiovascular, metabolic, and reproductive systems [1, 2]. Studies have shown that BPA undergoes hepatic metabolism via UDP-glucuronosyltransferase 1A1 (UGT1A1), which catalyzes its conjugation with glucuronic acid to form BPA glucuronide (BPAG). Due to its high water solubility, BPAG is predominantly eliminated via renal filtration and subsequent urinary excretion [3], thereby reducing its bioavailability and toxicity in humans.

BPA is capable of crossing the blood-brain barrier, and accumulating evidence suggests that BPA-induced neurodegenerative changes are manifesting at increasingly younger ages. Studies have demonstrated that BPA disrupts insulin signaling in the brain, promotes tau phosphorylation, and contributes to the accumulation of misfolded proteins [4], thereby inducing neurotoxicity and impairing neuronal development [5]. In neuronal cells, BPA activates the nuclear factor-kappa B (NF-κB) signaling pathway [6], leading to the production of reactive oxygen species (ROS), which in turn triggers the assembly and activation of the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome, ultimately resulting in neuroinflammation and neurodegeneration [7]. NF-κB, a key nuclear transcription factor, is a major regulator of inflammation, with the p65 subunit interacting with the gasdermin D (GSDMD) promoter region to activate GSDMD, subsequently triggering inflammatory responses [8]. The NLRP3 inflammasome is composed of NLRP3, ASC, and pro-caspase-1, which assemble to activate caspase-1 and promote the maturation of interleukin (IL)-1β and IL-18 [9]. ROS oxidize thioredoxin (TRX), causing the release of thioredoxin-interacting protein (TXNIP) and promoting TXNIP-dependent NLRP3 activation [10]. Activated caspase-1 also cleaves GSDMD to generate the pore-forming GSDMD-N fragment [11], which facilitates pyroptotic proinflammatory cytokine release [12].

Microglia, the resident macrophages of the human brain, serve as the primary line of defense in the central nervous system and play essential roles in neuronal development and synaptic pruning [13]. Upon BPA exposure, activated microglia generate excessive ROS, promoting oxidative stress and upregulating proinflammatory mediators, including tumor necrosis factor-alpha (TNF-α), IL-1β, and IL-6 [14]. The intracellular antioxidant defense system—comprising enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) functions to counteract ROS-induced oxidative damage [15]. BPA has been shown to activate the ROS/NLRP3/caspase-1 axis, leading to increased ROS production, elevated malondialdehyde (MDA) levels, and reduced SOD activity, thereby promoting oxidative injury and pyroptosis in osteocytes MLO-Y4 cells [11]. Furthermore, BPA increases MDA levels while suppressing GPx, GR, SOD, and glutathione (GSH) activities in hepatic cells, contributing to oxidative stress and hepatotoxicity [16]. These findings highlight the significant roles of neuroinflammation and oxidative stress in BPA-induced toxicity. Given that many phytochemicals possess antioxidant and neuroprotective properties, dietary intervention may offer a practical and accessible strategy to prevent or mitigate the health risks associated with BPA exposure.

Carnosic acid (CA), a polyphenolic diterpene compound isolated from the leaves of rosemary (Rosmarinus officinalis L.), is a major antioxidant constituent known for its anti-inflammatory [17], anti-obesity, anticancer, and neuroprotective properties [18]. CA acts as an electrophilic compound that protects neurons from oxidative stress and neurotoxicity, thereby reducing synaptic damage [19, 20]. Importantly, CA not only exerts systemic effects but also crosses the BBB, reaching the brain parenchyma to confer neuroprotection, particularly in the context of chronic neurodegenerative diseases [21]. Previous studies have shown that pretreatment with CA prevents 6-hydroxydopamine-induced motor deficits by enhancing antioxidant enzyme expression and reducing lipid peroxidation in the brain [20]. Additionally, CA delivered via a nanocarrier alleviates microglial-mediated neuroinflammation by inhibiting the production of proinflammatory cytokines such as IL-1β and TNF-α, thereby preventing glial-induced neuronal damage under chronic inflammatory conditions and ultimately improving cognitive function [22]. However, the molecular mechanisms underlying the neuroprotective effects of CA in human microglial cells (HMC3) under BPA exposure remain unclear. Therefore, this study aimed to investigate whether CA mitigates BPA-induced oxidative stress and neuroinflammation, potentially providing a preventive strategy against BPA-related neurotoxicity associated with environmental exposure.

Materials and Methods

Chemical

Carnosic acid (CA, purity ≧ 95%, CAS RN: 3650-09-7) was purchased from Cayman (Ann Arbor, MI). MEM medium, L-glutamine, trypsin-EDTA, and penicillin-streptomycin solution were obtained from Gibco Laboratory (Gaithersburg, MD). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). BPA (purity ≧ 99%, CAS No. 80-05-7), dimethyl sulfoxide (DMSO), protease inhibitor, phosphatase inhibitor, and sodium dodecyl sulfate (SDS) were purchased from Sigma Chemical Company (St. Louis, MO). Acrylamide/bis-acrylamide solution and RIPA buffer were from Biokit (Taiwan). Tris was purchased from Affymetrix (Cleveland, Ohio). PARP was purchased from Cell Signaling (Beverly, MA). Primary antibodies against NLRP3, TXNIP, ASC, cleaved caspase 1, GSDMD, IL-1β, and GR were purchased from GeneTex (San Antonio, TX). Primary antibodies against IL-6, IL-18, UGT1A1, GPx1, SOD2, and catalase were purchased from ABclonal (Danvers, MA). FKHR1 (FoxO1), ^Ser396^p-tau, nuclear factor erythroid 2-related factor 2 (Nrf2), p65, GAPDH, mouse anti-rabbit IgG HRP antibody, and m-IgGk BP HRP antibody were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Cell Culture

The Human Microglia Clone 3 cell line (HMC3 cells, #CRL-3304, RRID: CVCL_II76) was obtained from the American Type Culture Collection (ATCC, USA). HMC3 microglial cells were grown in Minimum Essential Medium (MEM) supplemented with 10% FBS, 2 mmol/L L-glutamine, 1.5 g/L sodium bicarbonate, 1.0 mmol/L sodium pyruvate, 1 × 10^5^ unit/L penicillin, and 100 mg/L streptomycin at 37 °C under a humidified atmosphere of 95% air and 5% CO_2_ [23].

Treatments

CA and BPA were dissolved in DMSO. HMC3 cells were plated at a density of 2 × 10^4^ cells per dish in 35-mm plastic tissue culture dishes, and the dishes were incubated until 90% confluence was reached. HMC3 cells were treated with 20 nM BPA and 1 µM CA for 3 h [24]. Cells treated with 0.1% DMSO alone were used as controls. Cells were treated with 100 µM buthionine-sulfoximine (BSO) for 18 h before BPA and CA treatment [25].

Cell Viability Assay

Cell viability was assessed using the MTT assays. HMC3 cells were seeded in 35-mm plastic tissue culture dishes at a density of 2 × 10^4^ cells per dish and incubated according to the experimental design. HMC3 cells were co-treated with 20 nM BPA in the presence or absence of 1 µM CA for 3 and 24 h. Cells treated with 0.1% DMSO alone served as the control group. After treatment, HMC3 cells were washed with phosphate-buffered saline (PBS) and incubated with MTT at a final concentration of 0.5 mg/mL in MEM medium at 37 °C for 2 h [26]. After removing the medium, the dishes were placed in isopropanol to dissolve the formazan crystals. Absorbance was measured at 570 nm by using an ELISA reader (Bio Rad, Japan). The value in the control cells was considered as 100%. The experiment was conducted in biological triplicates.

ROS Content Detection

2′,7′-Dichlorodihydrofluorescein Diacetate (DCFH-DA), a membrane-permeable compound, undergoes enzymatic conversion to the highly fluorescent 2′,7′-dichlorofluorescein (DCF) in the presence of ROS, facilitating the detection and quantification of ROS in cells [27]. For ROS detection, we use flow cytometry (DCFH-DA probes) and fluorescence imaging. HMC3 cells were treated with 20 nM BPA and 1 µM CA for 30 min. Cells were treated with 100 µM BSO for 24 h before BPA and CA treatment. Following treatment, the cells were washed with PBS and incubated with 10 µM DCFH-DA for 20 min at 37 °C to detect ROS. After dye incubation, the cells were washed with PBS to eliminate excess probe. Trypsin (200 uL) was applied for 2 min to detach the cells, and enzymatic activity was neutralized by adding 800 uL of culture medium. The cell suspension was centrifuged at 3,000 x g for 5 min, and the resulting cell pellet was resuspended in 500 µL PBS. Fluorescence signals reflecting ROS production were quantified using flow cytometry. In the meantime, we visualized green fluorescence alteration in the single-cell suspension under a fluorescence microscope (Leica, Germany) with excitation and emission wavelengths set to 485/520 nm. The results were expressed as a percentage relative to DCF fluorescence in the respective control cells. Intracellular ROS levels were quantified using ImageJ software, and all experiments were conducted in biological triplicates.

Western Blotting

After washing cells with cold PBS, the total protein content from HMC3 cells was harvested with RIPA lysis buffer (including 1% phosphatase inhibitor and 1% protease inhibitor). Lysates were cleared by centrifugation at 14,000 x g for 20 min at 4 °C. In addition, the nuclear protein extracts from HMC3 cells were collected using PBS and centrifuged at 2,000 x g for 5 min at 4 °C. The resulting cell pellet was resuspended in a hypotonic buffer containing 10 mmol/L HEPES, 10 mmol/L KCl, 1 mmol/L MgCl, 1 mmol/L EDTA, 0.5 mmol/L DTT, 4 ug/mL leupeptin, 20 g/mL aprotinin, 0.5% nonidet P-40, and 0.2 mmol/L phenylmethylsulfonyl fluoride (PMSF). The cell solution was centrifuged at 6,000 × g for 15 min at 4 °C, and the resulting cell pellet containing crude nuclear material was resuspended in a hypertonic buffer containing 10 mmol/L HEPES, 400 mmol/L KCl, 1 mmol/L MgCl, 1 mmol/L EDTA, 0.5 mmol/L DTT, 4 ug/mL leupeptin, 20 g/mL aprotinin, 10% glycerol, and 0.2 mmol/L PMSF. The nuclear extract solution was incubated on ice for 30 min and subsequently centrifuged at 6,000 × g for 15 min at 4 °C. Protein concentrations were then measured at 595 nm by using an ELISA reader (Bio-Rad, Japan). Ten micrograms of protein samples were separated on 7.5%, 10% or 12.5% SDS-PAGE. The protein is transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). The nonspecific binding sites on the membranes were blocked at 4 °C overnight with 50 g/L nonfat dry milk in 25 mM Tris/150 mM NaCl buffer, pH 7.4. The blots were then incubated with primary antibodies against PARP (CAS No. #142), NLRP3 (CAS No. GTX133569), TXNIP (CAS No. GTX 31592), ASC (CAS No. GTX 102474), cleaved caspase 1 (CAS No. GTX 101322), GSDMD (CAS No. GTX636896), IL-1β (CAS No. GTX74034), GR (CAS No. GTX114199), IL-6 (CAS No. A0286), IL-18 (CAS No. A1115), UGT1A1 (CAS No. A6186), GPx1 (CAS No. A1110), SOD2 (CAS No. A1340), catalase (CAS No. A11777), FoxO1 (CAS No. sc-374427), ^Ser396^p-tau (CAS No. sc-32275), Nrf2 (CAS No. sc-722), p65 (CAS No. sc-8008), or GAPDH (CAS No. sc-47724) overnight at 4 °C and were subsequently incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG HRP antibody (CAS No. sc-2357), and m-IgGk BP HRP antibody (CAS No. sc-516102). Primary antibodies were used at a 1:1000 dilution, and horseradish peroxidase-conjugated secondary antibodies were used at a 1:2000 dilution. The bands were detected by using an Immobilon ECL Ultra Western HRP substrate (Millipore, Bedford, MA). GAPDH was used as an internal control for cytoplasmic proteins, and PARP was used as an internal control for nuclear proteins. The experiment was conducted in biological triplicates.

Immunocytochemistry (ICC)

The method was assessed as in our previous study [28]. HMC3 cells were plated on 24-well plastic tissue culture dishes. After treatment, cells were washed twice with warm PBS and fixed with 10% formaldehyde (containing 0.2% sucrose) at 37 °C for 20 min. Cells were then permeabilized with 0.3% Triton X-100 and blocked with 3% BSA. After blocking, cells were incubated with primary antibody solution (including NLRP3 and cleaved-caspase 1) at 37 °C overnight, followed by incubating with secondary antibody-Alexa Fluor 488 conjugate (purchased from Thermo Fisher Scientific, Rockford, IL) at 37 °C for 2 h. Hoechst 33258 dye was used to stain the nuclei (purchased from Sigma-Aldrich, Co, St. Louis, MO). The images were detected by using a fluorescence microscope. The experiment was conducted in biological triplicates.

Enzyme-linked Immunosorbent Assay (ELISA)

HMC3 cells were plated in 35-mm plastic tissue culture dishes and treated with 20 nM BPA and 1 µM CA for 48 h. Cells treated with 0.1% DMSO alone served as the control group. After treatment, IL-1β levels in the culture medium were quantified by ELISA using a human IL-1β ELISA kit (VAL101, Valukine™ ELISA Kit, USA) according to the manufacturer’s instructions. The experiment was conducted in biological triplicates.

Animals and Treatments

C57BL/6J male mice aged 3 weeks (purchased from National Laboratory Animal Center, Taipei, Taiwan) were used in this study. The protocols for animal-related experiments were approved by the Institutional Animal Care and Use Committee of China Medical University (protocol no. CMUIACUC-2021-099-1). Mice were kept in polypropylene cages under a temperature-controlled room at 23 ± 1 °C with a 12-h light/dark cycle. Animals were fed with a chow diet and water in glass bottles ad libitum. After 2 weeks of adaptation, mice were randomly divided into four groups (n = 9): (1) control group: olive oil; (2) BPA group: 50 µg/kg BPA; (3) Low CA group: 50 µg/kg BPA + 5 mg/kg CA; (4) High CA group: 50 µg/kg BPA + 20 mg/kg CA [4]. The BPA and CA were dissolved in olive oil and administered by oral intubation once daily for 10 weeks. In week10, rats were anesthetized with isoflurane. The striatum was dissected out for the assays.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism version 8.0.2. Data were expressed as mean ± SD. Normality was assessed using the Shapiro-Wilk test. Differences among groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. All experiments were performed in biological triplicates. Statistical differences were considered significant when the p-value was less than 0.05.

Results

BPA Increased the Expression of NLRP3 Inflammasome-related Proteins

Studies have demonstrated that BPA activates the NLRP3 inflammasome [29]. Accordingly, in this study, Western blot analysis was employed to examine the effects of BPA at different concentrations and time points on inflammasome-related protein expression in HMC3 microglial cells. The results revealed that BPA at a concentration of 20 nM markedly increased protein levels of the inflammasome components NLRP3 and ASC to 98% and 90%, respectively, compared with the control group (Fig. 1A). Therefore, 20 nM BPA was selected for subsequent experiments. Moreover, BPA treatment for 3 h elicited the most pronounced induction of NLRP3 and ASC protein expression; therefore, this time point was selected for subsequent analyses (Fig. 1B). We then used it to further verify whether BPA activates NLRP3 inflammasome expression in HMC3 microglial cells. Consistently, treatment with 20 nM BPA for 3 h significantly enhanced NLRP3 fluorescence intensity (Fig. 1C). Therefore, a 20 nM BPA treatment for 3 h was used as the condition for the following experiments in this study.

Fig. 1BPA induced the protein expressions of NLRP3 and ASC in HMC3 cells. (A) HMC3 cells were treated with 0.1% DMSO or 2, 20 and 200 nM BPA for 3 h. (B) HMC3 cells were treated with 0.1% DMSO or 20 nM BPA for 1, 3 and 6 h. The protein expression was measured by Western blotting. GAPDH was used as the internal control. The protein level in control was regarded as 1. (C) HMC3 cells were treated with 0.1% DMSO or 20 nM BPA for 3 h. The green fluorescence indicated NLRP3. The nucleus is stained with Hoechst 33258 (blue). The merged images show the overlap of both fluorescences. The images shown are representative from three individual. Values are shown as mean ± SD (n = 3 biological triplicates). *p < 0.05, **p < 0.01, ***p < 0.001 compared to control group

CA Attenuated BPA-induced Expression of NLRP3 Inflammasome Proteins

After determining the optimal concentration and exposure time of BPA, we investigated whether CA was associated with attenuation of BPA-induced inflammasome-related protein expression in HMC3 microglial cells by analyzing NLRP3 and cleaved-caspase1 protein levels using ICC. The results showed that BPA significantly increased the fluorescence intensity of NLRP3 (Fig.2A) and cleaved caspase1 (Fig. 2B) compared with the control group, indicating increased expression of these inflammasome components. Notably, co-treatment with CA markedly reduced the expression levels of both NLRP3 (Fig. 2A) and cleaved caspase-1(Fig. 2B), thereby attenuating BPA-induced inflammasome-related protein expression. Furthermore, ELISA results showed that BPA induced the production of the proinflammatory cytokine IL-1β, whereas CA treatment significantly reduced IL-1β levels (Fig. 2C). Then, an MTT assay was performed to evaluate the effects of different BPA exposure durations on the viability of HMC3 cells. Cell viability indicated that treatment with 20 nM BPA for 3–24 h in the presence or absence of CA had no significant difference in cell viability compared with the control group (Fig. 2D). The results showed that BPA and CA at these exposure times were not cause cell death to HMC3 cells, however, CA improved the proteins expression of NLRP3 and IL-1β induced by BPA.

Fig. 2CA improved BPA-induced the proteins of NLRP3 and cleaved-caspase1 in BPA-treated HMC3 cells. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA for 3 h. (A and B) The green fluorescence indicated NLRP3 and cleaved-caspase1. The nucleus is stained with Hoechst 33258 (blue). The merged images show the overlap of both fluorescences. The images shown are representative from three individual. (C) HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 3 and 24 h. IL-1β production was measured by ELISA. (D) Cell viability was measured by MTT assay. The level in control was regarded as 100%. Values are shown as mean ± SD (n = 3 biological triplicates). **p < 0.01, ***p < 0.001 compared to control group. ^##^p < 0.01, ^###^p < 0.001 compared to BPA group

CA Attenuated BPA-induced Protein Levels in the Inflammasome, Pro-inflammatory Cytokines, and Inflammatory-Related Proteins

Previous reports have indicated that BPA induces NLRP3 inflammasome activation and elevates proinflammatory cytokine levels [12], thereby contributing to neuroinflammation [30]. The present study indicated that BPA treatment significantly upregulated the expression of TXNIP, NLRP3, ASC, cleaved-caspase-1, and GSDMD-N-terminal proteins compared with the control group, and then we explored the effect of CA. Notably, co-treatment with CA markedly suppressed the expression of these proteins, including TXNIP, NLRP3, ASC, cleaved-caspase-1, and GSDMD-N term levels by 70%, 62%, 37%, 50%, and 31%, respectively (Fig. 3A). Our results also showed that BPA significantly increased the protein levels of proinflammatory cytokines IL-6, IL-18, and IL-1β. However, CA co-treatment effectively attenuated the expression of IL-6, IL-18, and IL-1β by 40%, 57%, and 29%, respectively (Fig. 3), suggesting that CA was associated with reduced BPA-induced neuroinflammatory responses.

Fig. 3CA improved BPA-induced the protein expressions in inflammasome, pro-inflammatory cytokines and inflammatory related proteins in HMC3 cells. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 3 h. The protein expression was measured by Western blotting. GAPDH was used as the internal control. The protein level in control was regarded as 1. Values are shown as mean ± SD (n = 3 biological triplicates). *p < 0.05, **p < 0.01, ***p < 0.001 compared to control group. ^#^p < 0.05, ^##^p < 0.01, ^###^p < 0.001 compared to BPA group

Previous studies have shown that FoxO1 and its downstream target TXNIP promote ROS generation, thereby contributing to NLRP3 inflammasome activation [31]. Microglial activation enhanced NF-κB pathway signaling, leading to the release of proinflammatory cytokines and promoting NLRP3-mediated inflammatory responses [32]. Inflammatory responses promote tau phosphorylation in neuronal cells, thereby accelerating the progression of neurodegeneration [33]. In this study, we found that BPA significantly increased FoxO1 and ^Ser396^p-tau protein expression compared with the control group. However, co-treatment with CA significantly reduced the expression of both proteins of FoxO1 and ^Ser396^p-tau (Fig. 3C). Similar results were observed in nuclear proteins, BPA activated nuclear FoxO1 and p65 proteins, however, CA co-treatment alleviated the expression of these proteins (Fig. 3D). Therefore, CA was associated with attenuation of BPA-induced neuroinflammatory signaling by reducing FoxO1 and p65, as well as reducing the phosphorylation of tau, thereby mitigating NLRP3 inflammasome expression and proinflammatory cytokine production.

CA Attenuated BPA-induced Oxidative Stress

Previous studies have found that BPA induces a significant increase in intracellular ROS generation, leading to oxidative damage [34]. In this study, ROS levels in HMC3 microglial cells were measured using flow cytometry and the fluorescent probe DCFH-DA. After treating the cells with BPA in the presence or absence of CA, the results showed that BPA significantly increased ROS levels, which were subsequently reduced upon co-treatment with CA (Fig. 4A). Similar results were observed in ICC, HMC3 cells treated with BPA exhibited a significant increase in ROS expression (green fluorescence), which was diminished upon co-treatment with CA (Fig. 4B). These findings suggested that CA was associated with alleviation of BPA-induced oxidative stress in HMC3 microglial cells.

Fig. 4CA improved HMC3 cells against BPA-induced oxidative stress. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 0.5 h. (A) Reactive oxygen species (ROS) production was measured by flow cytometry. Fluorescence signals represent ROS production. (B) ROS generation was determined using DCF-DA assay. ROS level was visualized as DCF-DA green fluorescence. Values are shown as mean ± SD (n = 3 biological triplicates). *p < 0.05, ***p < 0.001 compared to control group. ^#^p < 0.05, ^###^p < 0.001 compared to BPA group

CA Restored the Reduction of Antioxidant-related Enzymes and Nrf2 Protein Levels Induced by BPA

Previous studies have shown that BPA enhances ROS generation, leading to the depletion of antioxidant enzymes, impairment of mitochondrial function, and subsequent alterations in cellular signaling pathways that ultimately result in cell death [35]. The antioxidant transcription factor Nrf2 regulates the expression of antioxidant-related proteins to mitigate oxidative damage [36]. The results of this study indicated that BPA significantly reduced the expression of antioxidant enzymes, including catalase, GR, SOD2, GPx1, and HO-1. However, co-treatment with CA significantly enhanced the protein levels of these antioxidant enzymes (Fig. 5A). Furthermore, BPA was found to reduce Nrf2 protein levels in the nucleus, whereas co-treatment with CA restored the Nrf2 protein (Fig. 5B). These findings suggested that CA was associated with restoration of BPA-reduced Nrf2 protein expression, leading to increased levels of antioxidant enzymes and enhanced antioxidant capacity.

Fig. 5CA reversed the BPA-decreased protein expressions in antioxidant enzymes and Nrf2 in HMC3 cells. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 3 h. The protein expression was measured by Western blotting. (A) GAPDH was used as the internal control. (B) PARP was used as the internal control. The protein level in control was regarded as 1. Values are shown as mean ± SD (n = 3 biological triplicates). **p < 0.01, ***p < 0.001 compared to control group. ^#^p < 0.05, ^##^p < 0.01, ^###^p < 0.001 compared to BPA group

CA Ameliorated the BPA-induced Reduction of Antioxidant Enzymes and the Detoxification Enzyme UGT1A1 Protein in BPA-treated Mice and HMC3 Cells

BPA is a substrate for the enzyme UGT1A1, which catalyzes its conjugation by glucuronidation, increasing its water solubility and facilitating its excretion [37]. We used Western blot analysis to examine the protein of UGT1A1 by CA in in vivo and in vitro. The results indicated that BPA resulted in a significant reduction of the UGT1A1 protein in the striatum of mice (Fig. 6A) and HMC3 microglial cells (Fig. 6B). Co-treatment with CA significantly increased the UGT1A1 protein. Moreover, consistent findings were observed in HMC3 microglial cells; CA significantly restored the SOD2 protein reduced by BPA in the striatum of mice (Fig. 6A). Although no significant difference in GPx1 protein expression was detected between the BPA-treated and control groups, CA co-treatment markedly increased the protein of GPx1 in the striatum of mice (Fig. 6A). These findings suggested that CA was associated with attenuation of BPA-induced reductions in antioxidant and detoxification enzymes, thereby enhancing intracellular antioxidant defense and promoting BPA metabolism in in vivo and in vitro.

Fig. 6CA reversed the proteins related to the antioxidant and detoxification enzyme in BPA-induced mice and HMC3 cells. (A) Mice were divided into 4 groups: control (olive oil 0.01 ml/g weight), BPA (50 µg/kg BPA), CA 5 (50 µg/kg BPA + 5 mg/kg CA), and CA 20 (50 µg/kg BPA + 20 mg/kg CA). (B) HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 3 h. The protein expression was measured by Western blotting. GAPDH was used as the internal control. The protein level in the control was regarded as 1. Values are shown as mean ± SD (n = 3 biological triplicates). ***p < 0.001 compared to control group. ^##^p < 0.01, ^###^p < 0.001 compared to BPA group

BSO Inhibited the Protective Effects of CA on the Oxidative Stress and NLRP3 Inflammasome Induced by BPA

To further confirm the antioxidant and anti-inflammatory role of CA in response to BPA, we utilized the GSH synthesis inhibitor BSO. The results showed that cells treated with BSO, CA no longer restored the antioxidant enzymes catalase, GR, SOD2, and GPx1 proteins, which were reduced by BPA (Fig. 7A), indicating that BSO treatment notably inhibited the antioxidant capacity of CA. We further analyzed the intracellular ROS generation, and the results showed that CA was unable to attenuate the BPA-induced ROS accumulation after BSO treatment in HMC3 microglial cells (Fig.7B). Evidence also suggests that elevated ROS levels can activate the NLRP3 inflammasome, thereby contributing to the development of neuroinflammation [30]. Our experimental findings revealed that BSO treatment blocked the ability of CA to modulate the expression of NLRP3 inflammasome-related proteins such as NLRP3, GSDMD-N terminal, and ASC, as well as the pro-inflammatory cytokines IL-18 and IL-1β that were induced by BPA (Fig. 7C). These findings suggested that CA was associated with attenuation of BPA-induced oxidative stress and prevented the subsequent expression of the inflammasome.

Fig. 7BSO blocked CA reversed the ROS and protein levels of antioxidant enzyme, NLRP3 inflammasome and inflammatory-related proteins in BPA-treated HMC3 cells. HMC3 cells were treated with 0.1% DMSO or 20 nM BPA in the presence or absence of 1 µM CA for 0.5 and 3 h. Cells were pretreated with 100 µM BSO for 18 h before treatment with CA and BPA. (A and C) The protein expression was measured by Western blotting. GAPDH was used as the internal control. The protein level in control was regarded as 1. (B) ROS production was measured by flow cytometry. Fluorescence signals represent ROS production. Values are shown as mean ± SD (n = 3 biological triplicates). *p < 0.05 compared to control group. ^#^p < 0.05, ^##^p < 0.01, ^###^p < 0.001 compared to BPA group. ^&^p < 0.05, ^&&^p < 0.01, ^&&&^p < 0.001 compared to BPA and CA group

Discussion

BPA is a widely recognized environmental endocrine-disrupting chemical that induces inflammatory and oxidative stress responses, leading to excessive ROS production and disruption of the cellular antioxidant defense system [38]. The resulting oxidative damage compromises the structural integrity of proteins and lipids, particularly in neural tissues, thereby contributing to neuroinflammation, apoptosis, and the development of behavioral and psychiatric disorders [39, 40]. Consequently, increasing attention has been directed toward identifying dietary compounds counteracting BPA-induced neurotoxicity. In this study, we found that BPA activated the NLRP3 inflammasome, leading to increased neuroinflammatory responses. Additionally, BPA exposure reduced the proteins of the antioxidant-related enzymes, the antioxidant transcription factor Nrf2, and the detoxifying enzyme UGT1A1, while simultaneously increasing intracellular ROS levels. Treatment with CA reversed BPA-suppressed antioxidant enzyme expression (Nrf2 and UGT1A1) and attenuated BPA-induced ROS production. These effects subsequently attenuated the NLRP3 inflammasome proteins, pro-inflammatory cytokines, and tau phosphorylation. This study revealed that CA mitigates BPA-induced oxidative stress and neuroinflammation. Notably, phosphorylated tau was used as a surrogate marker of neuronal tau pathology under neuroinflammatory conditions rather than as a direct indicator of microglial signaling. Accordingly, interpretations of neuronal tau pathology should be considered indirect and inferential.

BPA has been shown to induce inflammatory responses, particularly in the central nervous system, where neuroinflammation is a common pathological feature of many neurological disorders [41]. This neuroinflammatory response is primarily mediated by glial cell activation, particularly microglia [42]. Upon BPA exposure, microglia become activated and subsequently initiate the production of pro-inflammatory cytokines such as TNF-α and interleukins, along with inflammatory mediators including ROS and NOS, through activation of the NF-κB signaling pathway [43]. A study has shown that stimulation with lipopolysaccharide (LPS) upregulates the protein expressions of NLRP3 and caspase-1 in HMC3 cells, leading to increased production of the proinflammatory cytokines IL-1β and IL-18 [44]. Recent findings also reveal that green tea attenuates BPA-induced upregulation of NLRP3, ASC, cleaved caspase-1, GSDMD-N, IL-6, and IL-1β, thereby reversing BPA-induced microglial activation in the hippocampus and mitigating neuroinflammatory responses [45]. In this study, we found that BPA activated the NLRP3 inflammasome signaling pathway (Fig. 1), thereby enhancing inflammatory responses. Treatment with CA effectively reversed BPA-induced expression of NLRP3 inflammasome components (Figs. 2A and 3A) and reduced the expression of inflammation-associated proteins and pro-inflammatory cytokines (Fig. 3B). Future studies employing canonical inflammasome activation models, pathway-specific pharmacological or genetic approaches, and FoxO1-targeted interventions, such as siRNA and chromatin immunoprecipitation (ChIP) assays to assess FoxO1 binding to the TXNIP promoter. These experiments will help establish causality and more precisely delineate the roles of NLRP3/caspase-1 signaling and the FoxO1-TXNIP axis in BPA-induced neuroinflammation and CA-mediated neuroprotection. In addition, functional assays directly assessing caspase-1 enzymatic activity or pyroptotic membrane permeabilization (e.g., LDH release) will further strengthen and complement the evaluation of inflammasome activation.

The study indicated that BPA is primarily detoxified through glucuronidation, a phase II metabolic process catalyzed by UGT1A1 enzyme in the liver [37]. This detoxification pathway conjugates BPA with glucuronic acid, increasing its hydrophilicity and facilitating efficient excretion via urine or bile. Effective glucuronidation by UGT plays a critical role in reducing BPA bioavailability and mitigating its toxicological effects. This mechanism is particularly important in minimizing BPA-associated physiological damage, including its involvement in the pathogenesis of neurodevelopmental disorders such as autism spectrum disorders (ASDs) and attention-deficit/hyperactivity disorder (ADHD), as well as neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD), and endocrine-related conditions like polycystic ovary syndrome (PCOS) [46]. In the present study, BPA exposure significantly reduced UGT1A1 expression (Fig. 6), suggesting a potential disruption of detoxification pathways. Co-treatment with CA significantly restored UGT1A1 expression in both murine brain tissue and HMC3 microglial cells (Fig. 6). Although UGT1A1 upregulation is commonly associated with enhanced xenobiotic metabolism, these findings are suggestive rather than definitive of improved BPA clearance, as direct measurements of BPA metabolites were not performed. Nevertheless, CA mitigated BPA-induced downregulation of UGT1A1, indicating a potential role in modulating detoxification-associated signaling pathways.

Exposure to BPA induces pronounced oxidative stress, ultimately leading to neuronal cell damage [47]. BPA not only promotes excessive ROS generation but also suppresses the activity of antioxidant enzymes, including SOD, CAT, glutathione S-transferase (GST), GPx, and GR [48]. The resulting oxidative imbalance highlighted the need for exogenous antioxidant interventions to mitigate BPA-induced oxidative stress and protect against neuronal injury. A study has demonstrated that capsaicin upregulates Nrf2 and SIRT-1 expression in the liver of BPA-treated mice, while concomitantly decreasing malondialdehyde levels, enhancing antioxidant enzyme activity, and suppressing proinflammatory cytokine production, thereby mitigating BPA-induced metabolic dysregulation [49]. Recent studies have demonstrated the health-promoting properties of natural compounds, particularly flavonoids, which have considerable potential to alleviate oxidative stress and neurodegenerative diseases. For instance, diosmin, a naturally occurring flavonoid, has been shown to exert neuroprotective effects by attenuating BPA-induced oxidative stress and neuroinflammation [47]. Similarly, deacetyl epoxyazadiradione (DEA), a semi-synthetic derivative of epoxyazadiradione derived from neem (Azadirachta indica A. Juss) seeds, has demonstrated protective effects against BPA-induced toxicity in zebrafish larvae by alleviating oxidative stress and mitigating inflammatory responses [50]. In the present study, CA markedly upregulated the expression of antioxidant enzymes (Figs. 5A and 6A), restored Nrf2 activity (Fig. 5C), and reduced intracellular ROS levels (Fig. 4), collectively attenuating BPA-induced oxidative stress. However, these antioxidative and anti-inflammatory effects of CA were significantly diminished in cells pretreated with BSO (Fig. 7), suggesting that the antioxidant defense system plays a critical role in mediating the protective actions of CA. Nevertheless, behavioral and functional neurological outcomes were not assessed in this study, which limits direct evaluation of the physiological relevance of these findings. Future in vivo studies are therefore warranted to systematically evaluate the potency and efficacy of CA in mitigating BPA-induced inflammatory responses.

Conclusion

Taken together, these findings suggest that CA suppressed BPA-induced oxidative stress by reducing intracellular ROS generation, enhancing antioxidant enzyme expression, restoring Nrf2 expression, and upregulating the detoxification enzyme UGT1A1. These effects were associated with attenuation of NLRP3 inflammasome–related protein expression and downstream proinflammatory cytokine production.